The present invention relates to a liquid feeder for automatic culture apparatus, and more particularly to a liquid feeder for injection of a liquid such as a culture, buffer or enzyme solution into a culture vessel in an automatic culture apparatus in which biological tissues or cells are automatically cultured.
The technique of culturing biological tissues and cells represents an essential fundamental experimental process in various fields including the medical, biological, pharmaceutical and agricultural fields. However, the culture of biological tissues and cells over successive generations involve a technical difficulty, which prevents a stable strain being cultured from being obtained in practice. Thus, there has been a need for a procedure for culturing biological tissues and cells which enable a stabilized strain being cultured to be obtained. Recently, a culture technique in a gaseous environment within an incubator has been developed, and has enabled the culturing over successive generations of cells of a particular variety such as those of liver, neuron, pituitary gland which have been considered difficult to be cultured.
The culture over successive generations will be briefly summarized below. A given number of cells is diluted in a culture solution in the form of a suspension, which is injected into a culture vessel such as petri dish. The vessel is placed in an incubator which cultures the cells under a given gas atmosphere. After a predetermined period of time, the vessel is removed from the incubator and the number of growth of cells are counted under a microscope. When it is determined that the intended cells have grown over the full extent of the vessel, it is transferred to a strain-free clean bench and the culture solution in the vessel is withdrawn with a pipette and disposed. Subsequently, a buffer solution is injected into the vessel to clean the remaining cells, and then withdrawn for disposal with a pipette. The grown cells which attach to the bottom of the vessel are rendered freely removable therefrom by the injection of an enzyme solution such as trypsin and allowing the vessel to remain intact for a given period. After the predetermined period, the enzyme solution is withdrawn from the vessel with a pipette and disposed, and a culture solution is again injected into the vessel. The culture solution is repeatedly withdrawn and discharged through the pipette to cause an oscillation or agitation which enables the grown cells to be completely released from the bottom of the vessel and suspended in the culture solution. The cells in suspension are transferred into a centrifuge tube with a pipette, and placed in a centrifuge to separate the cells from the solution. Thereupon the cells attach to the bottom of the tube while the culture solution will be a decantered solution, which is disposed by tilting the tube. A culture solution is again injected into the centrifuge tube and is agitated by utilizing the withdrawing and discharging operation through the pipette to separate the cells from each other so that they are uniformly suspended in the culture solution within the centrifuge tube. Finally, the solution is distributed into a pair of culture vessels in an equal amount to complete one culture operation.
It will be recognized by those skilled in the art that the foregoing culture technique makes it necessary to remove the culture vessel out of the incubator and to expose it to the outer atmosphere in order to examine the growth of the tissues or cells under a microscope. This causes a sudden change in the culture conditions since the cells or tissues are placed out of a given environment maintained within the incubator including a gas atmosphere, temperature and humidity. This causes a delicate influence upon the tissues or cells being cultured and also involves an unavoidable contamination thereof by miscellaneous strains present in the atmosphere.
In addition, the various operations required for culturing over successive generations which should take place based on the results of observation with the microscope rely on a manual operation by an operator in the clean bench. This means that any slight difference in the various operations from operator to operator may have a direct influence upon the culturing result of the tissues or cells. Since the experience and skill of culturing technique varies from operator to operator, it is difficult to provide a standard procedure for the culturing technique, and this makes it impossible to obtain cultured tissues or cells of uniform quality. As a consequence, for different groups of researchers conducting a common study on the same theme, the conclusions reached may depend on the quality of the tissues being cultured. In extreme cases, the conclusions may be opposite to each other. Thus it will be seen that the reliability cannot be expected when the tissues or cells are cultured with the conventional technique.
It is generally accepted that it takes at least two years to train a skilled operator. As a result there exists, a continued demand for skilled operators. As a consequence, researchers often have to perform the culturing operation themselves rather than devotedly directing their efforts to their study.
In view of these considerations, the present invention is directed to an automatic culture apparatus capable of automatically performing the above described culturing operation. As a result, the present invention eliminates the contamination which may occur as a result of the exposure to the atmosphere, eliminates the influence of manual operations upon the cultured results and permits a standard and uniform procedure for the various culturing operations to be established.
An automatic culture apparatus meeting the above requirements must be provided with a liquid supply system or liquid feeder which supplies various liquids or solutions such as buffer, enzyme and culture solutions to the culture vessel or centrifuge tube. The liquid supply system must include a refrigerator for storing the buffer, enzyme or culture solution. This refrigerator cannot, however, be housed within an incubator having a limited capacity since a large quantity of such solutions is required. As a result, it is necessary to provide a liquid supply from outside the incubator to a given location within the incubator maintained under a predetermined environment. In addition, the entire liquid feeder must be sterilized before it is assembled with the automatic culture apparatus. If the feeder is disassembled before sterilization, the parts may be subjected to contamination by strains during the assembly which follows the sterilization step, adversely influencing the subsequent culturing operation. It is desirable, therefore, to design the feeder so that it can be sterilized without requiring the disassembly thereof. It is also desirable that the liquid feeder be designed such that it can be mounted to extend into the incubator so that it is easily detachable in order to permit its replacement by a fresh feeder whenever the stored solution or solutions are exhausted. In addition, a leakage of the atmosphere through the connection must be prevented.
After a buffer solution is injected into the vessel of the automatic culture apparatus, it becomes necessary to switch the supply from a buffer solution to an enzyme solution since it is desired to inject the enzyme solution into the same vessel. The conventional practice is to inject two different solutions selectively into the same vessel. This has been done utilizing a pair of separate injection pumps which are connected with supply tube of the respective solutions. The tubes have their discharge port located above the vessel, and the pumps are separately operated. However, the location of the discharge port of the respective supply tubes above the vessel was undesirable since residual solution sometimes dripped down from the port after the injection of the solution ceased resulting in a possible contamination of the vessel. Accordingly, the prior art technique was inadequate for a culturing procedure for which a high accuracy is required.
During the culturing operation, it is also required for the liquid feeder to provide a three dimensional movement of a single discharge port in order to inject the culture solution into a culture vessel and a centrifuge tube which are located at different sites. The conventional practice has been to use a pair of motors, one for vertical movement and the other for rotation. In order to control the termination or stop position of the vertical and rotational movements, there must be provided four limit switches in total, which resulted in a complex arrangement, which is also liable to malfunctioning by the failure of the limit switches.